This project involves the study of a rapidly emerging group of immune receptors. Many families of inhibitory immune receptors have recently been identified in both mice and humans. Interestingly, within each of these inhibitory families of receptors, there are proteins that have lost the inhibitory domains. Instead these receptors have gained a positively charged acid within their transmembrane domain, suggesting that they may interact with signal transduction chains and transmit positive signals. In this project, we study the signal transduction and biochemistry of both the positive and negative regulators of immune cell function. Through the study of the positive receptors, we and others demonstrated the association of some of these receptors with the novel signal transduction chain DAP12. Since then we have been characterizing the biochemistry of the the DAP12 signal transduction pathway. This work has included demonstration of the kinases involved in the early signaling of DAP12, delineation of the adaptors involved, and study of the regulation of these pathways. To facilitate these studies, we have developed and are utilizing, a reconstitution system that involves transfection of the receptor, DAP12, a kinase, and a reporter plasmid. This system allows for the analysis of various components of the pathway via co-transfection. In addition, biochemical analysis of the pathway has defined several substrates of DAP12-activated kinases. The exact roles of these substrates in mediating specific DAP12-driven responses is under investigation. In addition, to DAP12, we are beginning the study of DAP10, a second chain known to associate with receptors within NK cells and monocytes that is located just 130 bp from DAP12 on Chromosome 19. DAP10 contains a tyrosine based motif unique from that of DAP12. This motif (Y*xNM) suggests interaction with both the phosphatidylinositol 3 kinase (PI3K) and the adaptor Grb2. We are now preparing to dissect the signaling of DAP10 in an effort to fully understand the role of these chains within NK cells, monocytes and dendritic cells. Our studies of paired receptor systems has now largely shifted to the study of the Triggering Receptors Expressed on Myeloid cells (TREM). Our recent identification of TLT-1, a putative inhibitory receptor within the TREM cluster has defined the TREM as paired receptor system. TREM-1 has recently been shown to be involved in the amplification of signals leading to septic shock. TREM-2 has been reported to be involved in the maturation of dendritic cells. Together these data suggest the TREM are involved in the regulation of both the innate and adaptive immune response. What role, if any, TLT-1 plays in the regulation of the TREM is now under investigation. The pattern of expression of TLT-1 mirrors that of TREM-1, and we have demonstrated the ability of TLT-1 to become phosphorylated and recruit the protein phosphatase SHP-1. In addition, we have produced a fusion protein containing the extracellular portion of TLT-1 fused to the Fc region of human IgG. Using this soluble fusion protein as a probe we have begun to search for TLT-1 ligands. It is our feeling that these studies will uncover the role of TLT-1 in immune regulation allowing for the identification of drug targets within the TLT-1 pathway that may be important in the control of viral infection, autoimmunity, and malignancy.